Draft Genome Sequence of Bacillus halotolerans IcBac2.1, a Strain with Potential as a Phytopathogen Control Agent.

Several species of the genus Bacillus are used as plant growth-promoting bacteria. In particular, species of the subtilis group are known as good antagonists of phytopathogenic fungi. Here, we report the draft genome sequence of a rhizospheric Bacillus strain with promising abilities as a biocontrol agent.

[Genetic characterization of rice endophytic bacteria (Oryza sativa L.) with antimicrobial activity against Burkholderia glumae]. / Caracterización genética de bacterias endofíticas de arroz (Oryza sativa L.) con actividad antimicrobiana contra Burkholderia glumae.

The aim of the present study was to isolate, select and characterize endophytic bacteria in rice inhibiting Burkholderia glumae THT as well as to characterize the genetic diversity and virulence factors in strains of B. glumae and Burkholderia gladioli of rice. Rice plants were collected in 4 departments from the northern region of Peru, isolating endophytic bacteria, after tissue sterilization, at 30°C (48h) in Trypticase Soy Agar (TSA), evaluating the antimicrobial activity against B. glumae THT, production of siderophores, resistance of toxoflavine and partial sequencing of the 16S rRNA gene. Furthermore, B. glumae and B. gladioli were isolated in selective medium (pH 4.5) at 41°C/72h. Molecular identification was performed using BOX-PCR and sequencing of the 16S rRNA gene, in addition to the production of extracellular enzymes, motility tests and sensitivity/resistance to bactericides. One hundred and eighty nine (189) endophytic bacteria were isolated, and only 9 strains showed antimicrobial activity against B. glumae THT, highlighting Burkholderia vietnamiensis TUR04-01, B. vietnamiensis TUR04-03 and Bacillus aryabhattai AMH12-02. The strains produced siderophores and at least 55.5% were resistant to toxoflavin. Additionally, 17 strains were grouped into 9 BOX-PCR profiles, where 16 had similarity with B. glumae LMG2196T (100%) and 1 with B. gladioli NBRC 13700T (99.86%). High diversity was found according to geographical origin and virulence factors. In conclusion, strains of the genus Bacillus and Burkholderia are potential biocontrol agents against B. glumae.

Identification of the symbiosis island of Bradyrhizobium paxllaeri LMTR 21T.

The complete symbiosis island (SI) of Bradyrhizobium paxllaeri LMTR 21T, a mutualistic symbiont of the legume Phaseolus lunatus, was identified and analyzed. The SI was 646 kb in size, had lower G+C content than the genome average, and encoded not only nodulation and nitrogen fixation functions but also those for hydrogen uptake, vitamin and phytohormone biosynthesis, molybdenum transport, nonribosomal peptide synthesis, and type III secretion. Additionally, two divergent nodA genes were encoded in the SI.

A Genomotaxonomy View of the Bradyrhizobium Genus.

Whole genome analysis of the Bradyrhizobium genus using average nucleotide identity (ANI) and phylogenomics showed the genus to be essentially monophyletic with seven robust groups within this taxon that includes nitrogen-fixing nodule forming bacteria as well as free living strains. Despite the wide genetic diversity of these bacteria no indication was found to suggest that the Bradyrhizobium genus have to split in different taxa. Bradyrhizobia have larger genomes than other genera of the Bradyrhizobiaceae family, probably reflecting their metabolic diversity and different lifestyles. Few plasmids in the sequenced strains were revealed from rep gene analysis and a relatively low proportion of the genome is devoted to mobile genetic elements. Sequence diversity of recA and glnII gene metadata was used to theoretically estimate the number of existing species and to predict how many would exist. There may be many more species than those presently described with predictions of around 800 species in nature. Different arguments are presented suggesting that nodulation might have arose in the ancestral genus Bradyrhizobium.

The Type VI secretion system of Rhizobium etli Mim1 has a positive effect in symbiosis.

The Type VI secretion systems (T6SSs) allow bacteria to translocate effector proteins to other bacteria or to eukaryotic cells. However, little is known about the role of T6SS in endosymbiotic bacteria. In this work we describe the T6SS of Rhizobium etli Mim1, a bacteria able to effectively nodulate common beans. Structural genes and those encoding possible effectors have been identified in a 28-gene DNA region of R. etli Mim1 pRetMIM1f plasmid. Immunodetection of Hcp protein, a conserved key structural component of T6SS systems, indicates that this secretion system is active at high cell densities, in the presence of root exudates, and in bean nodules. Rhizobium etli mutants affected in T6SS structural genes produced plants with lower dry weight and smaller nodules than the wild-type strain, indicating for the first time that the T6SS plays a positive role in Rhizobium-legume symbiosis.

Nodule bacteria from the cultured legume Phaseolus dumosus (belonging to the Phaseolus vulgaris cross-inoculation group) with common tropici phenotypic characteristics and symbiovar but distinctive phylogenomic position and chromid.

Phaseolus dumosus is an endemic species from mountain tops in Mexico that was found in traditional agriculture areas in Veracruz, Mexico. P. dumosus plants were identified by ITS sequences and their nodules were collected from agricultural fields or from trap plant experiments in the laboratory. Bacteria from P. dumosus nodules were identified as belonging to the phaseoli-etli-leguminosarum (PEL) or to the tropici group by 16S rRNA gene sequences. We obtained complete closed genomes from two P. dumosus isolates CCGE531 and CCGE532 that were phylogenetically placed within the tropici group but with a distinctive phylogenomic position and low average nucleotide identity (ANI). CCGE531 and CCGE532 had common phenotypic characteristics with tropici type B rhizobial symbionts. Genome synteny analysis and ANI showed that P. dumosus isolates had different chromids and our analysis suggests that chromids have independently evolved in different lineages of the Rhizobium genus. Finally, we considered that P. dumosus and Phaseolus vulgaris plants belong to the same cross-inoculation group since they have conserved symbiotic affinites for rhizobia.

Nitrogen Fixation in Cereals.

Cereals such as maize, rice, wheat and sorghum are the most important crops for human nutrition. Like other plants, cereals associate with diverse bacteria (including nitrogen-fixing bacteria called diazotrophs) and fungi. As large amounts of chemical fertilizers are used in cereals, it has always been desirable to promote biological nitrogen fixation in such crops. The quest for nitrogen fixation in cereals started long ago with the isolation of nitrogen-fixing bacteria from different plants. The sources of diazotrophs in cereals may be seeds, soils, and even irrigation water and diazotrophs have been found on roots or as endophytes. Recently, culture-independent molecular approaches have revealed that some rhizobia are found in cereal plants and that bacterial nitrogenase genes are expressed in plants. Since the levels of nitrogen-fixation attained with nitrogen-fixing bacteria in cereals are not high enough to support the plant's needs and never as good as those obtained with chemical fertilizers or with rhizobium in symbiosis with legumes, it has been the aim of different studies to increase nitrogen-fixation in cereals. In many cases, these efforts have not been successful. However, new diazotroph mutants with enhanced capabilities to excrete ammonium are being successfully used to promote plant growth as commensal bacteria. In addition, there are ambitious projects supported by different funding agencies that are trying to genetically modify maize and other cereals to enhance diazotroph colonization or to fix nitrogen or to form nodules with nitrogen-fixing symbiotic rhizobia.

Draft Genome Sequence of Rhizobium sophoriradicis H4, a Nitrogen-Fixing Bacterium Associated with the Leguminous Plant Phaseolus vulgaris on the Coast of Peru.

The genome sequence of Rhizobium sophoriradicis H4, a nitrogen-fixing bacterium isolated from the common bean (Phaseolus vulgaris) in Peru, is reported here. The genome assembly revealed a 6.44-Mbp genome which was distributed into 95 contigs, with N50 and L50 values of 293 kbp and 9, respectively. The genome contained 6,312 coding sequence (CDS) genes and 52 RNA genes (49 tRNAs and 3 rRNAs).

Complete Genome Sequence of the Symbiotic Strain Bradyrhizobium icense LMTR 13T, Isolated from Lima Bean (Phaseolus lunatus) in Peru.

The complete genome sequence of Bradyrhizobium icense LMTR 13T, a root nodule bacterium isolated from the legume Phaseolus lunatus, is reported here. The genome consists of a circular 8,322,773-bp chromosome which codes for a large and novel symbiotic island as well as genes putatively involved in soil and root colonization.

Genome sequence of Bradyrhizobium sp. LMTR 3, a diazotrophic symbiont of Lima bean (Phaseolus lunatus).

Bradyrhizobium sp. LMTR 3 is a representative strain of one of the geno(species) of diazotrophic symbionts associated with Lima bean (Phaseolus lunatus) in Peru. Its 7.83 Mb genome was sequenced using the Illumina technology and found to encode a complete set of genes required for nodulation and nitrogen fixation, and additional genes putatively involved in root colonization. Its draft genome sequence and annotation have been deposited at GenBank under the accession number MAXC00000000.

Complete Genome Sequences of Eight Rhizobium Symbionts Associated with Common Bean (Phaseolus vulgaris).

We present here the high-quality complete genome sequences of eight strains of Rhizobium-nodulating Phaseolus vulgaris Comparative analyses showed that some of them belonged to different genomic and evolutionary lineages with common symbiotic properties. Two novel symbiotic plasmids (pSyms) with P. vulgaris specificity are reported here.

Draft genome sequence of Bradyrhizobium paxllaeri LMTR 21T isolated from Lima bean (Phaseolus lunatus) in Peru.

Bradyrhizobium paxllaeri is a prevalent species in root nodules of the Lima bean (Phaseolus lunatus) in Peru. LMTR 21T is the type strain of the species and was isolated from a root nodule collected in an agricultural field in the Peruvian central coast. Its 8.29 Mbp genome encoded 7635 CDS, 71 tRNAs and 3 rRNAs genes. All genes required to stablish a nitrogen-fixing symbiosis with its host were present. The draft genome sequence and annotation have been deposited at GenBank under the accession number MAXB00000000.

Genome Sequence of the Symbiotic Type Strain Rhizobium tibeticum CCBAU85039T.

Rhizobium tibeticum was originally isolated from root nodules of Trigonella archiducis-nicolai grown in Tibet, China. This species is also able to nodulate Medicago sativa and Phaseolus vulgaris The whole-genome sequence of the type strain, R. tibeticum CCBAU85039T, is reported in this study.

Candidatus Dactylopiibacterium carminicum, a Nitrogen-Fixing Symbiont of Dactylopius Cochineal Insects (Hemiptera: Coccoidea: Dactylopiidae).

The domesticated carmine cochineal Dactylopius coccus (scale insect) has commercial value and has been used for more than 500 years for natural red pigment production. Besides the domesticated cochineal, other wild Dactylopius species such as Dactylopius opuntiae are found in the Americas, all feeding on nutrient poor sap from native cacti. To compensate nutritional deficiencies, many insects harbor symbiotic bacteria which provide essential amino acids or vitamins to their hosts. Here, we characterized a symbiont from the carmine cochineal insects, Candidatus Dactylopiibacterium carminicum (betaproteobacterium, Rhodocyclaceae family) and found it in D. coccus and in D. opuntiae ovaries by fluorescent in situ hybridization, suggesting maternal inheritance. Bacterial genomes recovered from metagenomic data derived from whole insects or tissues both from D. coccus and from D. opuntiae were around 3.6 Mb in size. Phylogenomics showed that dactylopiibacteria constituted a closely related clade neighbor to nitrogen fixing bacteria from soil or from various plants including rice and other grass endophytes. Metabolic capabilities were inferred from genomic analyses, showing a complete operon for nitrogen fixation, biosynthesis of amino acids and vitamins and putative traits of anaerobic or microoxic metabolism as well as genes for plant interaction. Dactylopiibacterium nif gene expression and acetylene reduction activity detecting nitrogen fixation were evidenced in D. coccus hemolymph and ovaries, in congruence with the endosymbiont fluorescent in situ hybridization location. Dactylopiibacterium symbionts may compensate for the nitrogen deficiency in the cochineal diet. In addition, this symbiont may provide essential amino acids, recycle uric acid, and increase the cochineal life span.

Rhizobium favelukesii sp. nov., isolated from the root nodules of alfalfa (Medicago sativa L).

Strains LPU83T and Or191 of the genus Rhizobium were isolated from the root nodules of alfalfa, grown in acid soils from Argentina and the USA. These two strains, which shared the same plasmid pattern, lipopolysaccharide profile, insertion-sequence fingerprint, 16S rRNA gene sequence and PCR-fingerprinting pattern, were different from reference strains representing species of the genus Rhizobium with validly published names. On the basis of previously reported data and from new DNA-DNA hybridization results, phenotypic characterization and phylogenetic analyses, strains LPU83T and Or191 can be considered to be representatives of a novel species of the genus Rhizobium, for which the name Rhizobium favelukesii sp. nov. is proposed. The type strain of this species is LPU83T (=CECT 9014T=LMG 29160T), for which an improved draft-genome sequence is available.

Genomes of Candidatus Wolbachia bourtzisii wDacA and Candidatus Wolbachia pipientis wDacB from the Cochineal Insect Dactylopius coccus (Hemiptera: Dactylopiidae).

Dactylopius species, known as cochineal insects, are the source of the carminic acid dye used worldwide. The presence of two Wolbachia strains in Dactylopius coccus from Mexico was revealed by PCR amplification of wsp and sequencing of 16S rRNA genes. A metagenome analysis recovered the genome sequences of Candidatus Wolbachia bourtzisii wDacA (supergroup A) and Candidatus Wolbachia pipientis wDacB (supergroup B). Genome read coverage, as well as 16S rRNA clone sequencing, revealed that wDacB was more abundant than wDacA. The strains shared similar predicted metabolic capabilities that are common to Wolbachia, including riboflavin, ubiquinone, and heme biosynthesis, but lacked other vitamin and cofactor biosynthesis as well as glycolysis, the oxidative pentose phosphate pathway, and sugar uptake systems. A complete tricarboxylic acid cycle and gluconeogenesis were predicted as well as limited amino acid biosynthesis. Uptake and catabolism of proline were evidenced in Dactylopius Wolbachia strains. Both strains possessed WO-like phage regions and type I and type IV secretion systems. Several efflux systems found suggested the existence of metal toxicity within their host. Besides already described putative virulence factors like ankyrin domain proteins, VlrC homologs, and patatin-like proteins, putative novel virulence factors related to those found in intracellular pathogens like Legionella and Mycobacterium are highlighted for the first time in Wolbachia Candidate genes identified in other Wolbachia that are likely involved in cytoplasmic incompatibility were found in wDacB but not in wDacA.

Genome of Rhizobium leucaenae strains CFN 299(T) and CPAO 29.8: searching for genes related to a successful symbiotic performance under stressful conditions.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is the most important legume cropped worldwide for food production and its agronomic performance can be greatly improved if the benefits from symbiotic nitrogen fixation are maximized. The legume is known for its high promiscuity in nodulating with several Rhizobium species, but those belonging to the Rhizobium tropici "group" are the most successful and efficient in fixing nitrogen in tropical acid soils. Rhizobium leucaenae belongs to this group, which is abundant in the Brazilian "Cerrados" soils and frequently submitted to several environmental stresses. Here we present the first high-quality genome drafts of R. leucaenae, including the type strain CFN 299(T) and the very efficient strain CPAO 29.8. Our main objective was to identify features that explain the successful capacity of R. leucaenae in nodulating common bean under stressful environmental conditions. RESULTS: The genomes of R. leucaenae strains CFN 299(T) and CPAO 29.8 were estimated at 6.7-6.8 Mbp; 7015 and 6899 coding sequences (CDS) were predicted, respectively, 6264 of which are common to both strains. The genomes of both strains present a large number of CDS that may confer tolerance of high temperatures, acid soils, salinity and water deficiency. Types I, II, IV-pili, IV and V secretion systems were present in both strains and might help soil and host colonization as well as the symbiotic performance under stressful conditions. The symbiotic plasmid of CPAO 29.8 is highly similar to already described tropici pSyms, including five copies of nodD and three of nodA genes. R. leucaenae CFN 299(T) is capable of synthesizing Nod factors in the absence of flavonoids when submitted to osmotic stress, indicating that under abiotic stress the regulation of nod genes might be different. CONCLUSION: A detailed study of the genes putatively related to stress tolerance in R. leucaenae highlighted an intricate pattern comprising a variety of mechanisms that are probably orchestrated to tolerate the stressful conditions to which the strains are submitted on a daily basis. The capacity to synthesize Nod factors under abiotic stress might follow the same regulatory pathways as in CIAT 899(T) and may help both to improve bacterial survival and to expand host range to guarantee the perpetuation of the symbiosis.

Complete Genome Sequence of Bradyrhizobium sp. Strain CCGE-LA001, Isolated from Field Nodules of the Enigmatic Wild Bean Phaseolus microcarpus.

We present the complete genome sequence of Bradyrhizobium sp. strain CCGE-LA001, a nitrogen-fixing bacterium isolated from nodules of Phaseolus microcarpus. Strain CCGE-LA001 represents the first sequenced bradyrhizobial strain obtained from a wild Phaseolus sp. Its genome revealed a large and novel symbiotic island.

Bradyrhizobium viridifuturi sp. nov., encompassing nitrogen-fixing symbionts of legumes used for green manure and environmental services.

Symbiotic nitrogen-fixing bacteria, commonly called rhizobia, are agronomically important because they can provide significant amounts of nitrogen to plants and help in recovery of impoverished soils and improvement of degraded environments. In recent years, with advances in molecular techniques, several studies have shown that these bacteria have high levels of genetic diversity, resulting in taxonomic reclassifications and descriptions of new species. However, despite the advances achieved, highly conserved 16S ribosomal genes (16S rRNA) do not elucidate differences between species of several genera, including the genus Bradyrhizobium. Other methodologies, such as multilocus sequence analysis (MLSA), have been used in such cases, with good results. In this study, three strains (SEMIAs 690T, 6387 and 6428) of the genus Bradyrhizobium, isolated from nitrogen-fixing nodules of Centrosema and Acacia species, without clear taxonomic positions, were studied. These strains differed from genetically closely related species according to the results of MLSA of four housekeeping genes (dnaK, glnII, gyrB and recA) and nucleotide identities of the concatenated genes with those of related species ranged from 87.8 % to 95.7 %, being highest with Bradyrhizobium elkanii. DNA-DNA hybridization (less than 32 % DNA relatedness) and average nucleotide identity values of the whole genomes (less than 90.5 %) indicated that these strains represented a novel species, and phenotypic traits were determined. Our data supported the description of the SEMIA strains as Bradyrhizobium viridifuturi sp. nov., and SEMIA 690T ( = CNPSo 991T = C 100aT = BR 1804T = LMG 28866T), isolated from Centrosema pubescens, was chosen as type strain.